Nucleic acid shearing or fragmentation provides the a first step in several embodiments for constructing nucleic acid libraries, as well as, embodiments for hybridization of target nucleic acids on solid supports, for example microarrays. These embodiments benefit from controlled shearing forces to provide increased efficiency in fragmentation and subsequent analysis. Nucleic acid, for example DNA or RNA, fragmentation are the focus of next-generation sequencing platforms such as those by 454 (Roche Molecular, Inc.), SOLiD (Applied Biosystems), and Solexa (Illumina, Inc.). These platforms each have different embodiments for nucleic acid fragmentation that determine different parameters such as efficiency of fragmentation, fragmentation time, fragment length distribution range, and quality of fragments generated. For example, double-stranded DNA may be accompanied by single-stranded (denatured) DNA, or damaged (depurinated) DNA. Furthermore, there are several applications of next-generation sequencing that would benefit from higher throughput that could be achieved by reducing the time needed to process each sample.
There are different methods for nucleic acid fragmentation. Nucleic acids can be fragmented chemically by enzymatic digestion, for example, by DNasel. Nucleic acids can be fragmented mechanically, such as by hydrodynamic shearing or sonication. Mechanical fragmentation can occur by several methods known in the art, including shearing of DNA by passing it through the narrow capillary or orifice (Oefner et al., Nucleic Acids Res. 1996; Thorstenson et al., Genome Res. 1995), for example a hypodermic needle, sonicating the DNA, such as by ultrasound (Bankier, Methods Mol. Biol. 1993), grinding in cell homogenizers (Rodriguez L V. Arch Biochem Biophys. 1980), for example stirring in a blender, or nebulization. Mechanical fragmentation results, in some methods, in double strand breaks within a DNA molecule. Sonication is used widely for random fragmentation of nucleic acids for sequencing library or microarray probe preparations. A variety of instruments available on the market can provide sonication for nucleic acid preparation. Sonication may also be performed using any convenient approach, e.g., with a multi-tip sonicator or using acoustic sound waves. A Microplate Sonicator® (Misonix Inc.) may be used to partially fragment the DNA. Such a device is described in U.S. Patent Publication No. 2002/0068872. Other examples of sonicators for nucleic acid fragmentation are Vialtweeter or Sonotrode. Another acoustic-based system that may be used to fragment DNA is described in U.S. Pat. Nos. 6,719,449, and 6,948,843 manufactured by Covaris Inc. U.S. Pat. No. 6,235,501 describes a mechanical focusing acoustic sonication method of producing high molecular weight DNA fragments by application of rapidly oscillating reciprocal mechanical energy in the presence of a liquid medium in a closed container, which may be used to mechanically fragment the DNA. An exemplary configuration of such an instrument is shown at FIG. 4 illustrating a concave transducer focusing the acoustic energy through a water bath to a focal zone in the sample liquid contained in the sample vessel. In various embodiments, the focused acoustic energy can have a frequency of 1.1 MHz with 400 Watts of power applied (100 Watt maximum at 20% duty cycle), on a volume of approximately 2 millimeters in diameter by 6 millimeters in height (depth by length). Sonication parameters (such as power, duty cycle, and cycles per burst can be adjusted in the process recipe through software settings. The sonication region shape and volume is hardware dependent and can be modified with hardware changes. Purified nucleic acids can be amplified prior to or after a fragmentation step.
The shearing of a nucleic acid molecule in a liquid medium is achieved through the hydrodynamic action of the liquid on the molecule itself. When a velocity gradient exists within the liquid medium, the shear stresses produced by the elongational components of the flow result into an aligning and extensional action on the nucleic acid molecules along the direction of the shear stresses.
When the applied hydrodynamic action (tensile forces, bending moments, ect) builds up to exceed the intrinsic strength of the polymeric chain, a breakage in the chain will result, giving rise to two fragments, each shorter than the original. In general, since the hydrodynamic action applied to the nucleic acid molecule is proportional to its length, it is increasingly difficult to shear a fragment of a nucleics acid as it becomes shorter and shorter. The shear stress “tau”τ in the fluid giving rise to the hydrodynamic action on the polymeric chain can be expressed according to the following formula:
      τ    xy    =      μ    ·          (                                    ∂            u                                ∂            y                          +                              ∂            v                                ∂            x                              )      where μ“mu” is the viscosity of the liquid medium and du/dy, and dv/dx, the velocity gradients within the flow field. In order to enhance the shearing action and thus decrease size fragment and increase shearing throughput, the shear stress can be increased by increasing the viscosity of the liquid medium or by increasing the velocity gradients within the flow field. High concentration glycerol solutions are normally used to increase the viscosity of the liquid medium by several orders of magnitude compared to pure water. In addition, mechanisms such as sonication, can be used to produce stronger flow fields than otherwise achievable through more basic mechanical devices. For example, the minimum fragment size obtained in a Hydroshear instrument, where the liquid is force through a small orifice, is of the order of hundreds of base pair. On the other hand, fragments as short as tens of base pairs can be easily obtained with a sonicator thanks to the violence of the flow generated by ultrasonic cavitation. Taking into account the shear being the sum of elongation and rotation to cause stretching and tumbling to produce scission. This results in 5′-CpG-3′ preferential cleavage on double-stranded DNA (Grokhovsky, Mol. Bio., 2006). Typical parameters for optimizing nucleic acid fragmentation are sonication parameters (bursts per cycle, intensity, and duty cycle), process temperature, buffer viscosity, sample volume, nucleic acid amount, sample vessel size and material, buffer ionic strength, and nucleic acid purification method.
Varying lengths of fragments can be provided depending on the sequencing platform. For example, the Illumina 1 G sequencing platform requires the sonication of pure DNA to generate 100-300 bp pieces for fragment libraries, and of chromatin to get fragments in 200-700 bp range for ChIP-sequencing, using i.e. 250 Sonifier (Branson) or Bioruptor (Diagenode AS). Another example, SOLiD used sonication for fragment library preparation to generate size ranges of 60 to 90 base pair fragments from purified nucleic acids. This can be achieved with a Covaris, Inc. S2 sonicator fragmenting the nucleic acid for 40 minutes at maximum setting for power and frequency. Exploiting such instruments at maximum capacity for long periods of time, such as those needed to process each nucleic acid sample, can accelerate instrument aging visible as decline in instrument performance. Further, 40 minute fragmentation cycles (like in the SOLiD protocol) limit the throughput for sequencing instruments by extending the time necessary for gene library generation. In addition, sonication of purified nucleic acids is carried out in glycerol or other viscous liquids to increase the friction on the nucleic acids. However, the glycerol is then separated from the nucleic acid fragments. This process requires chemical extraction and can reduce the recovery of the nucleic acid fragments. Furthermore, long fragmentation cycles under high power settings increases the probability and extent of damage and denaturation of the purified nucleic acids (Milowska et al., Biomolecular Engineering 2007). This can be attributed to cavitation induced by sonication. Cavitation can collapse microbubbles, produce microjets, or produce shock waves in the sample liquid, as well as, generating strong flow, localized temperature rise, production of free radicals, for example H and OH (Fuciarelli et al, Free Radical Biology & Medicine, 1995).
It is desirable to provide a method for preparing nucleic acid fragments from a sample of purified nucleic acid that reduces the length of fragmentation time. It is desirable to provide a method for preparing nucleic acid fragments from a sample of purified nucleic acid that avoids glycerol or other viscous liquids and fragments the nucleic acid in an aqueous solution. It is desirable to increase the recovery of fragmented nucleic acids by reducing the power settings of sonicators or reducing the loss of nucleic acid fragments to post-fragmentation separation. It is also desirable to improve sequencing results by reducing the bias of sonication toward certain fragment sizes or fragment types (as opposed to the randomness of nebulization or hydroshearing). The present invention provides these desired results with a method for preparing nucleic acid fragments from a sample of purified nucleic acid by adding particles to the sample and sonicating the suspension. It is counterintuitive that adding particles to the sample would provide the desired fragmentation because the increase in viscosity tends to stop the mechanism for fragmentation at certain point by greatly reducing the molecules spatial turnover in the focal point of sonication. Therefore, the desirable results of the present invention are not predictable based on current understanding of nucleic acid fragmentation.
Particles have been used with sonication to lyse cells, see for example U.S. Pat. No. 6,440,725 describing a cartridge for cell lysis using beads and an ultrasonic transducer. However, the application of particles in cell lysis via sonication cannot achieve nucleic acid fragmentation because of the cell lysate present. Furthermore, nucleic acid fragmentation is contrary to the goal of cell lysis for detecting low copy of DNA targets in large volume sample, i.e. for diagnostics. To detect low-copy nucleic acids targets after cell lysis it is desirable to have the extracted DNA in high molecular weight form, as excessive degradation by i.e. oversonication can dramatically reduce the average DNA fragment length below the length of amplicon, thus greatly reducing the sensitivity of such PCR-based diagnostic methods.
It is also desirable to provide shearing of material that contains some nucleic acid, for example chromatic with DNA and proteins. It is also desirable to provide shearing of other long polymers that are not organic in nature. The present teachings can be expanded to material for fragmentation that includes any substance including nucleic acid, for example chromatin that includes a nucleic acid and protein, and any long polymer, other than nucleic acid where applications require shearing of the polymer for further processing.